Project Summary Nearly 4 million Americans over 65 are living with calcific aortic valve disease (CAVD). CAVD pathogenesis is an active biological process that is untreatable by cholesterol metabolism modifying agents. There are currently no successful biological targets or therapeutic agents that specifically target CAVD. Aortic valve cusp homeostasis and pathogenesis is regulated by complex and poorly understood interactions between resident surface valve endothelial cells (VEC) and underlying valve interstitial cell (VIC). While almost all research has focused on VIC, which cannot be isolated from VEC that surround them and faithfully reveal the biology of aortic valves and pathobiology of CAVD, this research project aims to unravel the roles of VIC, VEC and their interactions, at cell and molecule levels, for valve homeostasis and CAVD. Our preliminary data using new valve lineage specific genetic animal models show that activation of NF?B (a master gene mediating inflammation) in VEC and VIC, or inaction of Notch1 (a major regulator of cell fate and behavior) in VEC only results in CAVD in mice. Further, our innovative 3D in vitro culture of VEC identifies that NF?B activation in VEC promotes VEC undergo endothelial-to-mesenchymal transformation and generate a novel mesenchymal progenitor T-VIC that calcifies matrix. These exciting new results motivate the hypothesis that NF?B-Notch1 control valve homeostasis and CAVD pathogenesis via prevention or generation of these T-VIC. This proposal will be tested in three Specific Aims. First we will elucidate the role of Notch-NFkB in VEC homeostasis and CAVD related pathogenesis (Aim 1). Then we will elucidate how T-VIC affect VEC and/or VIC phenotypes in 3D co-culture in novel CAVD-related conditions (Aim 2). Aim 3 will evaluate the efficacy of NF?B-Notch1 signaling rebalancing on CAVD initiation and progression in vivo via reduction of T-VIC. The completion of this project will generate significant information regarding intercellular regulation of aortic valve homeostasis and the CAVD pathogenic process. By revealing the unique phenotypic signatures of participation VEC and VIC, our study will also identify and motivated new molecular candidates that specifically target the cell specific characteristics of the CAVD process for potential diagnostic and therapeutic benefit.